PROJECT SUMMARY . Despite the well-characterized consequences of disuse, we have a limited understanding of the early changes in the molecular environment that influence rehabilitation efforts in men and women. We propose a 2-phase, randomized, clinical trial that includes 7-days of unilateral leg disuse (Phase 1), immediately followed by 14- days of bilateral leg rehabilitation (Phase 2). We will recruit middle-aged men and women; a historically neglected research demographic who present with a largely youthful phenotype, but are at risk of accelerated disuse atrophy. ! In Phase 1, we will explore the sex-specific effects of skeletal muscle disuse and characterize subjects most-and least-susceptible to disuse atrophy. In male and female volunteers, single-leg muscle atrophy will be induced using an established knee-brace/disuse protocol. We will obtain skeletal muscle biopsies to characterize the sex-specific, molecular signature of skeletal muscle disuse, while highlighting differences in traditional morphologic and functional outcomes. ! In Phase 2, we will map the early molecular time-course of rehabilitation in men and women and determine if disused and healthy muscle respond similarly to an exercise / rehabilitation intervention. Sex-specific volunteer cohorts will complete: i) a structured bilateral, resistance-exercise rehabilitation protocol, or ii) a passive, ambulatory recovery (Control). We will obtain muscle biopsies after 0, 48, 96 h of rehabilitation to characterize the early time course of recovery of molecular transducers of disuse. These early, pre-clinical molecular changes will be supported by traditional morphologic, body composition and muscle function outcomes. This project will address critical knowledge gaps that limit the efficacy of current strategies to restore muscle health following periods of disuse. Current strategies, while well intentioned, are largely inconsistent with the practice of evidence-based medicine and place a financial and human resource burden on our health care delivery system. By characterizing changes in the molecular, morphologic and functional landscape of skeletal muscle during disuse and rehabilitation and reposing our RNASeq data within the Gene Expression Omnibus (GEO) website, this study may serve as the foundation for future, targeted studies of skeletal muscle disuse in clinical populations with comorbid conditions.